Bottom Line:
Transduced cells were labelled with the nucleoside analogue 5-ethynyl-2'-deoxyuridine (EdU) to detect cells progressing through S phase.Hits were identified using high-content imaging and statistical analysis and confirmed with vectors using two different promoters (CMV and EF1α).The screen demonstrates the reliability, versatility and utility of our screening platform, and identifies novel cell cycle/proliferative activities for a number of genes.

ABSTRACTIn response to the growing need for functional analysis of the human genome, we have developed a platform for high-throughput functional screening of genes overexpressed from lentiviral vectors. Protein-coding human open reading frames (ORFs) from the Mammalian Gene Collection were transferred into lentiviral expression vector using the highly efficient Gateway recombination cloning. Target ORFs were inserted into the vector downstream of a constitutive promoter and upstream of an IRES controlled GFP reporter, so that their transfection, transduction and expression could be monitored by fluorescence. The expression plasmids and viral packaging plasmids were combined and transfected into 293T cells to produce virus, which was then used to transduce the screening cell line. We have optimised the transfection and transduction procedures so that they can be performed using robotic liquid handling systems in arrayed 96-well microplate, one-gene-per-well format, without the need to concentrate the viral supernatant. Since lentiviruses can infect both dividing and non-dividing cells, this system can be used to overexpress human ORFs in a broad spectrum of experimental contexts. We tested the platform in a 1990 gene pilot screen for genes that can increase proliferation of the non-tumorigenic mammary epithelial cell line MCF-10A after removal of growth factors. Transduced cells were labelled with the nucleoside analogue 5-ethynyl-2'-deoxyuridine (EdU) to detect cells progressing through S phase. Hits were identified using high-content imaging and statistical analysis and confirmed with vectors using two different promoters (CMV and EF1α). The screen demonstrates the reliability, versatility and utility of our screening platform, and identifies novel cell cycle/proliferative activities for a number of genes.

pone-0020057-g002: Workflow for the screening of the MCF-10A cells.The left-hand panel shows plate manipulations and cell treatments for each day of the screen. The right-hand panel shows a representative image of a scanned field as a pseudo-colour overlay (top) of individual scanning channels (bottom) shown individually for the boxed area (DAPI – blue, GFP – green, EdU – red, scale bar = 50 µM). Blue lines in the enlargements encircle the object (nucleus) area selected by the scanning algorithm. See Materials and Methods for further details.

Mentions:
The virus generated from the 1990 expression clones was used to assess the ability of the overexpressed ORFs to induce increased proliferation in the MCF-10A cells after EGF removal. In preliminary experiments in the 96-well microplate format we determined the optimal assay conditions that significantly reduced cell proliferation but did not significantly reduce cell viability after exposure to the virus. Under these conditions cells not exposed to virus undergo 2–3 division cycles after EGF withdrawal before depleting the media and arresting in the G1 phase of the cell cycle. As summarized in Figure 2, cells were plated at 2000 cells per well in complete media (containing EGF and 5% v/v horse serum) on Day 0, and transduced on Day 1. Medium without EGF and containing 1% v/v serum was used for subsequent volume top-up and a Day 2 medium change. On Day 4, cells were pulsed for 2 h with the nucleotide analogue EdU which is incorporated into the nuclear DNA of cells in S phase. Cells were then fixed and processed for high-content imaging by staining with DAPI to define nuclei and measure DNA content, and by cross-linking EdU with the fluorescent label Cy5 to mark S phase nuclei. Plates were then scanned on a high-content imager, and individual nuclei scored for DAPI, GFP and Cy5 fluorescence, based on cumulative total (DAPI, Cy5) or average (GFP) nuclear pixel intensity.

pone-0020057-g002: Workflow for the screening of the MCF-10A cells.The left-hand panel shows plate manipulations and cell treatments for each day of the screen. The right-hand panel shows a representative image of a scanned field as a pseudo-colour overlay (top) of individual scanning channels (bottom) shown individually for the boxed area (DAPI – blue, GFP – green, EdU – red, scale bar = 50 µM). Blue lines in the enlargements encircle the object (nucleus) area selected by the scanning algorithm. See Materials and Methods for further details.

Mentions:
The virus generated from the 1990 expression clones was used to assess the ability of the overexpressed ORFs to induce increased proliferation in the MCF-10A cells after EGF removal. In preliminary experiments in the 96-well microplate format we determined the optimal assay conditions that significantly reduced cell proliferation but did not significantly reduce cell viability after exposure to the virus. Under these conditions cells not exposed to virus undergo 2–3 division cycles after EGF withdrawal before depleting the media and arresting in the G1 phase of the cell cycle. As summarized in Figure 2, cells were plated at 2000 cells per well in complete media (containing EGF and 5% v/v horse serum) on Day 0, and transduced on Day 1. Medium without EGF and containing 1% v/v serum was used for subsequent volume top-up and a Day 2 medium change. On Day 4, cells were pulsed for 2 h with the nucleotide analogue EdU which is incorporated into the nuclear DNA of cells in S phase. Cells were then fixed and processed for high-content imaging by staining with DAPI to define nuclei and measure DNA content, and by cross-linking EdU with the fluorescent label Cy5 to mark S phase nuclei. Plates were then scanned on a high-content imager, and individual nuclei scored for DAPI, GFP and Cy5 fluorescence, based on cumulative total (DAPI, Cy5) or average (GFP) nuclear pixel intensity.

Bottom Line:
Transduced cells were labelled with the nucleoside analogue 5-ethynyl-2'-deoxyuridine (EdU) to detect cells progressing through S phase.Hits were identified using high-content imaging and statistical analysis and confirmed with vectors using two different promoters (CMV and EF1α).The screen demonstrates the reliability, versatility and utility of our screening platform, and identifies novel cell cycle/proliferative activities for a number of genes.

ABSTRACTIn response to the growing need for functional analysis of the human genome, we have developed a platform for high-throughput functional screening of genes overexpressed from lentiviral vectors. Protein-coding human open reading frames (ORFs) from the Mammalian Gene Collection were transferred into lentiviral expression vector using the highly efficient Gateway recombination cloning. Target ORFs were inserted into the vector downstream of a constitutive promoter and upstream of an IRES controlled GFP reporter, so that their transfection, transduction and expression could be monitored by fluorescence. The expression plasmids and viral packaging plasmids were combined and transfected into 293T cells to produce virus, which was then used to transduce the screening cell line. We have optimised the transfection and transduction procedures so that they can be performed using robotic liquid handling systems in arrayed 96-well microplate, one-gene-per-well format, without the need to concentrate the viral supernatant. Since lentiviruses can infect both dividing and non-dividing cells, this system can be used to overexpress human ORFs in a broad spectrum of experimental contexts. We tested the platform in a 1990 gene pilot screen for genes that can increase proliferation of the non-tumorigenic mammary epithelial cell line MCF-10A after removal of growth factors. Transduced cells were labelled with the nucleoside analogue 5-ethynyl-2'-deoxyuridine (EdU) to detect cells progressing through S phase. Hits were identified using high-content imaging and statistical analysis and confirmed with vectors using two different promoters (CMV and EF1α). The screen demonstrates the reliability, versatility and utility of our screening platform, and identifies novel cell cycle/proliferative activities for a number of genes.